The goals of this proposal (outlined in bold below) are to address central questions regarding four broad areas of peroxisome biogenesis and import pathways for peroxisomal matrix and membrane proteins, and the assembly and import of peroxisomal membrane proteins is a prerequisite for matrix protein import. Recent focus in the field has been on the import of matrix proteins. While much progress has been witnessed along this forefront, we know very little about the earliest stages of peroxisome biogenesis, including the origin of lipids, membranes and membrane proteins required for this organelle's growth. Are these generated de novo, as was believed until recently, or is there a role for the endoplasmic reticulum (ER) in this process? Contrary to the expectation of the model that all peroxisomes arise by budding and fission from pre-existing peroxisomes we recently found two Peroxins that are associated with distinct vesicular structures that are not peroxisomes. The first aim addresses the role of vesicle-associated, AAA-family proteins, Pex1p and Pex6p in peroxisome biogenesis. Because so little is known about peroxisomal membrane protein biogenesis and this is critical for an understanding of peroxisome biogenesis as a whole, our second aim is to define the role of Peroxins in the import of proteins into the peroxisomal membrane. The third goal addresses the quantitative and dynamic interactions between matrix peroxisomal targeting signals (PTSs), their mammalian receptors and docking proteins on the peroxisomal membrane, and the role of chaperones in modulating these interactions. This is to obtain a biochemical understanding of the early stages of peroxisomal matrix protein import, for which the genetics have already uncovered most of the players, but the biochemistry is still lagging. We will define the functions of key Peroxins in matrix protein import-specifically in receptor-ligand interactions, receptor docking to peroxisomes, and receptor release from the peroxisome to the cytosol. Our last goal is to address the role of ubiquitination (or a ubiquitin- like modification) in protein degradation, targeting or new functions during peroxisome biogenesis. We hope to define the role of a ubiquitin- conjugating enzyme, Pex4p, in peroxisome biogenesis.